Over 20 million women suffer from urinary tract infections (UTI) annually in the U.S., and 25-35% of these patients will suffer from recurrent infections, including approximately 900,000 patients, mostly women, who will suffer from highly recurrent (greater than 3 episodes per year) UTI. Antimicrobial treatment of UTIs has resulted in increasing antimicrobial resistance among uropathogenic E. coli (UPEC) to first-line empiric therapies, such as trimethoprim-sulfamethoxazole, and even to broad-spectrum fluoroquinolones. Those with frequent recurrent UTI must often resort to long-term prophylactic antimicrobial therapy, which in turn selects for more resistance. Alternative treatment options are desperately needed. Over the past two decades, elucidation of bacterial pathogenic pathways in UPEC has revealed that the mannose-binding tip adhesin protein of type 1 pili, FimH, is an essential virulence factor, and thus a novel therapeutic target for the prevention and treatment of UTI. With this knowledge, investigators have identified and developed ?-D- mannose derivatives, called ?mannosides,? as FimH antagonists that are efficacious in blocking FimH function, including the abilities of FimH to mediate biofilm formation and UPEC adherence to mannosylated receptors on bladder epithelial cells. Using an interdisciplinary approach that blends medicinal chemistry, microbiology, and pharmacology, much progress has been made to optimize these potent, orally available mannosides that attenuate acute virulence, and treat existing infections in established murine models of UTI. Importantly, mannosides are effective at treating antimicrobial-resistant clinical strains, including an ST131 ESBL ?Superbug? strain, in vivo. Mannosides can even potentiate therapy with an antimicrobial that UPEC are otherwise clinically resistant to, by preventing bacterial invasion into host tissue, thereby exposing UPEC to host immune responses and high levels of first-line antimicrobials that concentrate in the urine. This proposal is focused on continuing the lead optimization of mannosides from the Phase I SBIR project to identify a clinical candidate drug for human clinical trials of UTI therapy. Development efforts will focus on the optimization of C- linked mannosides and O-mannoside pro-drugs with improved drug-like properties, including increased metabolic stability and bioavailability. In vitro biological assays will be used to screen our lead candidates for potency. The in vivo pharmacokinetics (PK) and efficacy of our best lead mannoside FimH antagonists will then be evaluated in mice. Our models are relevant to human UTI, and allow evaluation of mannosides for prevention and treatment of acute, recurrent and chronic UTI. Together with separate, but complementary, preclinical studies funded by our industry partner, our proposed experiments will allow us to identify a clinical candidate mannoside drug to move forward to investigational new drug (IND) status. The ultimate goal of this project is to deliver a first-in-class anti-virulence FimH mannoside antagonist drug into human clinical trials, as a new oral antimicrobial-sparing therapeutic for millions of women suffering from UTIs.